Drug interactions with the in vivo synthesis of thromboxane and prostacyclin

The biosynthesis and metabolic degradation of thromboxane and prostacyclin are briefly described with particular emphasis on the peculiarities of the enzymes involved. This is of great importance for the understanding of this system and for proper interpretation of experimental data. The requirements for adequate methodology in studies designed to assess the in vivo synthesis of those prostanoids are discussed. The characteristics of the thromboxane-prostacyclin system in normals are presented in detail with particular emphasis on those facets of importance for interpretation of literature data, like the diurnal variation, the large interindividual variation etc. The present status of knowledge about the involvement of this system in various cardiovascular diseases as well as the interaction of drugs of various types with the in vivo synthesis of those prostanoids is reviewed in detail.     

The source of thromboxane and prostaglandins in experimental inflammation.

Although cyclo-oxygenase products have been detected at inflammatory sites the tissue of origin remains uncertain. Inflammatory exudates were collected from rats 4, 6, 8, 12 or 24 h after subcutaneous implantation of carrageenin-impregnated sponges. Concentrations of the cyclo-oxygenase products prostaglandin E2 (PGE2), 6-oxo-PGF1 alpha and thromboxane B2 (TXB2) in inflammatory exudates and serum (obtained from blood clotted at 37 degrees C) were measured by specific radioimmunoassays. TXB2 concentrations in exudates increased to about 100 ng ml-1 at 8 h but decreased to less than 20 ng ml-1 after 24 h. PGE2 concentrations increased from 4-12 h and remained between 80 and 120 ng ml-1 from 12-24 h. 6-oxo-PGF1 alpha had the same time course as that of PGE2 but concentrations were approximately one third of PGE2 values. TXB2 concentrations in serum from thrombocytopaenic rats were less than 5% of control values. Thrombocytopaenia did not affect TXB2, PGE2 or 6-oxo-PGF1 alpha concentrations or total leukocyte numbers in inflammatory exudates. Methotrexate-induced neutropaenia did not affect serum TXB2 concentrations but cyclo-oxygenase products (including TXB2) in 6 h inflammatory exudates were reduced by 60-95%. Colchicine (1.0 mg kg-1 s.c.) prevented leukocyte accumulation in sponge exudates and this was accompanied by a reduction in TXB2, PGE2 and 6-oxo-PGF1 alpha concentrations at 6 h. These results indicate that platelets are the source of TXB2 in clotting blood but do not contribute to cyclo-oxygenase activity in experimental inflammation. The results also suggest that migrating leukocytes are the major source of thromboxane and to a lesser degree prostaglandins in acute 6 h inflammatory exudates.     

Effects of naproxen on the in vivo synthesis of thromboxane and prostacyclin in man

Summary The effect of a single oral dose of 500 mg naproxen on the synthesis in vivo of thromboxane A₂ and prostacyclin was studied in healthy volunteers. The synthesis of the prostanoids was assessed by measuring the urinary excretion of the metabolites 2,3-dinor-TxB₂ and 2,3-dinor-6-keto-PGF₁, respectively, using stable isotope dilution assays based on gas chromatography — mass spectrometry.Naproxen caused significant inhibition of the excretion of both metabolites for about two days. The reduction of the thromboxane metabolite was more pronounced (75% inhibition) than that of the prostacyclin metabolite (about 50% inhibition).The data support the idea that naproxen causes reversible inhibition of cyclooxygenase.     

Effect of morphine on protein synthesis in synaptosomes and mitochondria of mouse brain in vivo

Summary The effect of morphine on incorporation of¹⁴C-leucine into proteins of synaptosomes and mitochondria of mouse brain was studied in vivo. Morphine significantly decreased the uptake of¹⁴C-leucine into the synaptosomes by about 20%. It did not affect the uptake into the mitochondria, however. The effect was dose-dependent and could be prevented by naloxone. Barbital, on the other hand, depressed the incorporation of¹⁴C-leucine into mitochondria more than into synaptosomes, thus showing a different pattern of inhibitory effects on protein metabolism. The depressing effect of morphine on uptake of¹⁴C-leucine into the synaptosomes in vivo is probably due to inhibition of axoplasmic transport of proteins, and is an effect of higher specificity than that of barbital.The skilful technical assistance of Mrs. I. Wübbolt is gratefully acknowledged. I am indebted to the Endo Laboratories Inc., Garden City, N. Y., for a generous gift of naloxone.     

Effect of antioxidants on the synthesis of prostaglandins, prostacyclin and thromboxane in different layers of the kidneys of old rats

The effects of L-tocopherol and synthetic antioxidants on synthesis of prostaglandins, thromboxane B2 and prostacyclin in different layers of the kidney against the background of chronic administration of polyene antibiotics (amphotericin B, levorin, nystatin) were studied in the experiments on aged rats. It was concluded that synthetic antioxidants regulate synthesis of these biologically active substances and also are able to protect the organ from the damaging effect of polyene antibiotics.     

Interactions of thromboxane A2 analogs and prostaglandins in isolated dog arteries

Interactions of ONO3708, a thromboxane (TX) A2 analog, and epithio-methano-TXA2 (sTXA2) or prostaglandins (PGs) were investigated in helical strips of dog cerebral, coronary, renal, and mesenteric arteries. In these arterial strips sTXA2 (10(-10) to 10(-7) M) produced a dose-dependent contraction, whereas ONO3708 up to 10(-6) M failed to contract the arteries but antagonized the contractile response to sTXA2. The inhibition tended to be greater in renal arteries than in the other arteries. Contractions induced by PGF2 alpha, PGE2, and PGD2 were also suppressed by treatment with low concentrations (3 X 10(-9) and 10(-8) M) of ONO3708. The attenuations of the response to sTXA2, PGF2 alpha, PGE2, and PGD2 did not appreciably differ. Norepinephrine-induced contractions were not influenced by ONO3708 up to 2 X 10(-7) M. On the other hand, relaxant responses to PGI2 of cerebral and renal arteries were not reduced by ONO3708. Prostaglandin H2 produced a transient contraction followed by a relaxation in cerebral and renal arteries. The contractile response was abolished by 10(-7) M ONO3708, and the relaxation was potentiated. It may be concluded that ONO3708 selectively antagonizes the vasoconstrictor action of TXA2, its analogs, and PGs but does not alter the action of vasodilator PGs. At least in part, sTXA2, PGF2 alpha, PGE2, and PGD2 appear to share the same receptive site responsible for vascular contraction.     

DNA microarray analysis of cannabinoid signaling in mouse brain in vivo

To identify novel genes involved in cannabinoid receptor-mediated signaling, we used cDNA microarrays to detect changes in mRNA expression in the forebrains of mice 12 h after they were given a single intraperitoneal dose of the naturally-occurring Cannabis sativa alkaloid Delta(9)-tetrahydrocannabinol (Delta(9)-THC) or the synthetic cannabinoid receptor agonist (R)-(+)-2,3-dihydro-5-methyl-3-[(morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-yl-1-naphtalenylmethanone mesylate [R(+)-WIN 55,212-2]. Of approximately 11,000 genes from a mouse brain cDNA library that were probed, 65 showed altered (increased or decreased at least 2-fold) expression after exposure to Delta(9)-THC, 41 after exposure to R(+)-WIN 55,212-2, and 20 genes after exposure to both drugs. Genes affected similarly by Delta(9)-THC and R(+)-WIN 55,212-2 were considered likely to reflect cannabinoid receptor activation, and expression of the protein products of two such genes not previously implicated in cannabinoid signaling-melanocyte-specific gene-related gene 1 (MRG1) and hexokinase 4 (glucokinase, GK)-was measured by Western blotting and immunohistochemistry. Western blots showed approximately 2-fold increases in the levels of both proteins in mouse forebrain. Immunohistochemistry revealed preferential localization of MRG1 to cerebral blood vessels and of GK to hypothalamic neurons. These findings suggest that MRG1 and GK are cannabinoid-regulated genes and that they may be involved in the vascular and hypothalamic effects of cannabinoids, respectively.     

Kinetic properties of the accumulation of 3H-raclopride in the mouse brain in vivo

Summary The kinetic properties of the accumulation of ³H-raclopride, a selective dopamine (DA) D-2 receptor antagonist, in mouse striatum in vivo was examined under various experimental conditions. The accumulation in striatum was saturable in contrast to that in cerebellum, which linearily increased with the dose. The specific binding of ³-Hraclopride in the striatum, defined as the difference in the accumulation in striatum and cerebellum 30 min after the injection was completely inhibited by the D-2 receptor antagonists spiperone and (-I-)-butaclamol [but not (–)butaclamol] and the DA receptor agonist N-n-propylnorapomorphine. The mean B _max value of the specific binding was 40.7 ± 2.8 pmol/g tissue and the mean apparent K _D value, based on the dose injected, was 87.8 ± 11.5 nmol/kg i. v. (18 different experiments). Pretreatment of the mice with a single injection of reserpine 4 h or 3 days beforehand reduced the apparent K _D value which in part seemed to be due to the decreased concentration of synaptic DA. Similarly, -butyrolactone injected immediately before raclopride reduced the apparent KD value, whereas amfonelic acid and (–)-amphetamine increased the observed K _D values. These findings indicate competition between endogenous DA and raclopride for the D-2 receptors. Both reserpine and -butyrolactone increased the apparent B _max value by about 50% which indicates a receptor pool of DA for which raclopride does not compete.Send offprint requests to S. B. Ross at the above address     

The benzazepine, SCH 23390, inhibits 3H-NPA binding in mouse brain in vivo

The fact that SCH 23390, a selective dopamine (DA) D1 antagonist, blocks the effects of D2 agonists suggests a functional coupling of D1 and D2 receptors. Therefore, the binding of SCH 23390 to D2 receptors was investigated in vivo using 3H-N-n-propylnorapomorphine (NPA), a D2 agonist, and 3H-spiperone and 3H-raclopride, both D2 antagonists. SCH 23390 failed to inhibit 3H-spiperone or 3H-raclopride binding; however, SCH 23390 was relatively potent in inhibiting 3H-NPA binding. These results suggest that (some) antidopaminergic effects of SCH 23390 may result from antagonism of a D2 agonist conformation of the D2 receptor.     

Regional uptake of neurotoxic and nontoxic amino acids in vivo by the infant mouse brain

The acidic amino acids, glutamate, cysteate and homocysteate. destroy neurons in the arcuate nucleus of the hypothalamus when administered subcutaneously to infant mice. but alanine. a neutral amino acid, does not, The neurotoxicity of glutamate, cysteate and homocysteate was confirmed in this study. It was further demonstrated that glutamate accumulates in the arcuate nucleus after the administration of glutamate but not after cysteate or homocysteate administration. This rules out the possibility that the neurotoxicity of the latter two compounds is mediated by the conversion in vivo of these compounds to glutamate. It supports the thesis that the similar neurotoxic manifestations of these compounds stem from the similar molecular structure they share. Alanine accumulated in the arcuate and ventromedial nuclei of the hypothalamus and, to a lesser extent, the medial nucleus of the thalamus but had no neuropathologic effects in any brain region. The failure of alanine to damage arcuate neurons is interpreted as evidence that, because of its dissimilar molecular structure. it either does not react or reacts very differently from the acidic neuroloxic amino acids at some receptor locus on neural membranes.     

Drug effects on developing mouse brain protein synthesis in vitro

Drugs known to produce facilitated learning and memory in experimental animals were tested for direct effects on protein synthesis in brain cells obtained from the cerebral cortex of mice. These drugs did not alter tritiated amino acid uptake into intracellular pools or in vitro protein synthesis when tested over a wide range of doses. The mechanism of facilitatory drug action did not appear related to direct effects on amino acid uptake or protein synthesis.     

Effects of prostaglandins and thromboxane analogues on bullock and dog iris sphincter preparations.

1 The bullock iris sphincter was contracted by low concentrations of prostaglandin E2 (PGE2), 16, 16-dimethyl PGE2 and 17,18,19,20-tetranor-16-p-chlorophenoxy PGE2. Other compounds with thromboxane-like actions, for example 11,9-epoxymethano PGH2, were also potent spasmogens, ZK 36374, a stable carbacyclin, was a partial agonist on the PGE-sensitive system of this tissue. 2 The thromboxane antagonist, EP 045, had little effect on the action of PGE2 and 16,16-dimethyl PGE2 on the bullock iris. 3 The dog iris sphincter was sensitive to PGF2 alpha but not to PGE2 and 11,9-epoxymethano PGH2. 4 16,16-dimethyl PGE2 had very low activity on the dog iris in contrast to its high activity on the bullock iris. The reverse was found with the 17,18,19,20-tetranor-16-m-trifluoromethylphenoxy analogue of PGF2 alpha (ICI 81008). This indicates a considerable selectivity of action of the two analogues. 5 The results are discussed in relation to the existing knowledge of prostanoid receptors.     

The polyamine synthesis inhibitor alpha-difluoromethylornithine is neuroprotective against N-methyl-D-aspartate-induced brain damage in vivo.

We examined the ability of alpha-difluoromethylornithine (DFMO), an inhibitor or ornithine decarboxylase, the rate limiting enzyme for polyamine biosynthesis, to protect the brain of the perinatal rat from N-methyl-D-aspartate (NMDA)-induced brain damage. Treatment of the rat pups with DFMO administered either by i.p. injection (500 mg/kg x 2) or through the milk of the mother (2% solution in mother's drinking water) significantly reduced, by 48 and 62%, respectively, the brain damage produced by intrastriatal NMDA injection. We conclude that activation of polyamine synthesis may mediate part of the neurotoxic action of NMDA.     

In vivo effect of methylmercury on protein synthesis in brain and liver of the rat

Rats were given daily sc injections of methylmercury chloride, 10 mg/kg for 7 consecutive days. The manifestation of the neurological syndrome in the rat and the accumulation of mercury in rat tissues resembled the observations of previous investigators. The incorporation in vitro of [¹⁴C]leucine into brain protein began to decrease during the latent period of intoxication and declined to 56% of the control values at the symptomatic period. The incorporation of [¹⁴C]leucine into liver protein was also inhibited to a similar extent at the symptomatic period following a remarkable stimulation at the early stage after the onset of administration of methylmercury. The impairment of protein synthesis in the brain and liver at the symptomatic period was confirmed by the incorporation in vivo of a ¹⁴C-labeled amino acid mixture into proteins of these tissues. The decrease in the [¹⁴C]leucine incorporation in the liver of poisoned rats was largely affected by nutritional deficiency due to decreased food intake, but that in the brain resulted from the direct effect of methylmercury on this tissue.     

Effects of imidazoline receptor ligands on monoamine synthesis in the rat brain in vivo

This study was designed to assess the effects of imidazoline drugs on putative presynaptic imidazoline receptors modulating brain monoamine synthesis in vivo. The accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used as a measure of the rate of tyrosine and tryptophan hydroxylation in various brain regions of naive rats and after irreversible α₂-adrenoceptor inactivation with EEDQ (1.6 mg/kg, i.p., 6 h). Clonidine (1–3 mg/kg), moxonidine (1–10 mg/kg) and rilmenidine (10 mg/kg) (mixed I₁/α₂ agonists) decreased dopa and 5-HTP synthesis in the cerebral cortex (14%–81%), hippocampus (27%–84%) and/or striatum (29%–56%), but these inhibitory effects were abolished in N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)-treated rats. Similarly, the stimulatory effect of efaroxan (mixed I₁/α₂ antagonist; 10 mg/kg) on dopa synthesis in the cortex (77%) and hippocampus (57%) was abolished by EEDQ. The selective I₁-ligand 2-endo-amino-3-exo-isopropylbicyclo-heptane (AGN-192403; 5–10 mg/kg) did not modify dopa or 5-HTP synthesis in any brain region in naive or EEDQ-treated rats. Idazoxan (mixed I₂/α₂ antagonist; 20 mg/kg) increased dopa synthesis in the cortex (111%) and hippocampus (87%), but the stimulatory effects were abolished by EEDQ. Moreover, idazoxan and efaroxan decreased 5-HTP synthesis in the cortex (12%–34%) and hippocampus (30%–34%) in a manner sensitive to blockade by the 5-HT_1A receptor antagonist WAY 100135. The selective I₂-ligands 2-(2-benzofuranyl)-2-imidazoline (2-BFI; 20 mg/kg) and 2-styryl-2-imidazoline (LSL 61122; 10 mg/kg) did not alter the synthesis of dopa or 5-HTP in the cortex or hippocampus. In striatum, 2-BFI (1–20 mg/kg) dose-dependently decreased dopa synthesis (ED₅₀: 5.9 mg/kg), reduced dopamine levels (6%–36%) and increased those of its metabolites DOPAC (15%–95%) and HVA (24%–74%). The inhibitory effect of 2-BFI on dopa/dopamine synthesis in striatum remained unchanged after alkylation of imidazoline receptors with isothiocyanatobenzyl imidazoline (IBI; 60 mg/kg, 6 h) or blockade of these receptors with 2-(2-ethyl 2,3-dihydro-2-benzofuranyl)-2-imidazole (KU-14R; 7–20 mg/kg). Therefore, most imidazoline drugs modulated the synthesis of brain monoamines through interaction with α₂-adrenoceptors or 5-HT_1A receptors. The results do not provide functional evidence for the existence of presynaptic imidazoline receptors regulating the synthesis of monoamines in the rat brain. Received: 4 January 1999 / Accepted: 12 April 1999 / Published online: 2 July 1999